PROJECT SUMMARY CD4+ Foxp3+ regulatory T cells (Tregs) are key players in preventing lethal autoimmunity and deleterious tissue inflammation. Diverse functions of Tregs involved in both immunological and non- immunological processes have been demonstrated. However, how Treg functional specification is coupled to their differentiation remains poorly understood. Unanswered key questions include: How a balanced Treg differentiation program is established? What is the biological significance of this balance? How Treg?s function is interfered by an interrupted Treg differentiation? Without this knowledge, the understanding of Treg physiology and therapeutic applications is incomplete. In our preliminary studies, we have found that two TCF/LEF family transcription factors (TFs), TCF1 and LEF1, act redundantly to control Treg homeostatic differentiation and function. We observed that gradient expression of TCF1 and LEF1 was associated with Treg differentiation status. Importantly, we found that stage-specific expression of TCF1 and LEF1 was required for Treg?s primary function in preventing autoimmunity. Our further analyses supported a central role of the IL-2?STAT5 signaling cascade in TCF1/LEF1-mediated Treg?s stability and function. Therefore, our data suggested a new model whereby TCF1 and LEF1 drive Tregs to a specific differentiation state permissive for their stability and suppressive function. Applying our established animal models and other key techniques, the objectives of this application are: to define a core TF network to establish a dynamic balance of Treg differentiation (Aim 1); to determine the role of TCF1+ LEF1+ Treg subset in suppressing incipient autoimmunity (Aim 2); and to explore the underlying mechanisms by which TCF1/LEF1 regulates Treg stability (Aim 3). Successful completion of these aims will significantly advance our understanding of how TCF/LEF TFs regulate Treg differentiation, stability and function and provide new guidelines to improve Treg-based therapies to treat autoimmune and other complex diseases.